Project Overview: This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the non-replicating, drug-tolerant Mycobacterium tuberculosis bacteriocidal assay.

In this assay, we treated Vero E6 cells with compounds selected as "hits" in the M. tuberculosis (AID 488890) assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.034 uM to 20 uM. Following 72 hours of treatment, relative viable cell number was determined using a Cell Titer Glo (Promega). Each plate contained 64 replicates of vehicle treated cells which served as negative controls.

Protocol: Cell Culture: Vero E6 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide. Cells were passaged as needed, harvested from flasks using 0.25% trypsin-EDTA and maintained for no more than 20 passages.Compound Dosing/Plating: Compounds and carrier control (DMSO) were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume). Cell Plating: Twenty uL of complete growth medium containing 3000 cells were dispensed per well. Plates were incubated at 37 C, 5% CO2 for 72h prior to endpoint detection.Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature for 10 min. Twenty-five muL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader in luminescence mode with an integration time of 0.1 s.Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.

Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active". If the % viability at all doses was >70%, the compound was defined as "Inactive". Scoring: SRBCSC uses a three-tier scoring system where scores of 0-40 apply to primary screen data, 41-80 indicates confirmatory screen data, and 81-100 is reserved for confirmatory data on resynthesized compounds. Inactive compounds at any screening level receive a score of 0. In this confirmatory screen, "Active" compounds were scored based on CC50 results on a tier of 40-80 with "Inactive" compounds scoring 0.